The present invention, in some embodiments thereof, relates to coupling agents and, more particularly, but not exclusively, to the preparation and use of novel coupling agents that can be beneficially utilized in the syntheses of substances such as peptides and oligonucleotides.
Therapeutic peptides, or pharmaceutical peptides, take an ever-growing cut in the active pharmaceutical ingredients (API) market, particularly as, for example, antibiotics, hormones, immunomodulators, anti-angiogenesis agents, therapeutic agents for treating CNS and other neurological disorders, analgesics, anti-obesity drugs, and as therapeutic agents for treating immune disorders such as allergy, asthma, hemophilia, anemia and autoimmune diseases [for a review, see, Loffet, A., “Peptides as Drugs: Is There a Market?”, 2002, J. Peptide Sci. 8, 1]. According to a Frost & Sullivan report, there are currently more than 40 marketed peptide drugs worldwide, around 270 peptides in clinical phase testing, and about 400 in advanced preclinical phases. Peptides represent 1% of all total API with a market share estimated at US$300-500 M per year and an annual growth rate of 15-25%. It is expected that the market will double in the next few years, when generic and recently approved new chemical entities enter the market.
However, although peptides have enormous therapeutic potential, their widespread use has been limited by several restrictive technical factors. Today, manufacturing companies face the unprecedented challenge of producing hundred kilograms to tons quantities of complex peptides. Such a massive production typically uses expensive and complex modern technologies, rendering peptide manufacture difficult and cost-inefficient as compared with other “small-molecule” pharmaceuticals. Large-scale manufacturing and purification of peptides in a bioactive form can therefore be a limiting step in the commercialization of peptide-based drugs.
A key step in the peptide production process is the controlled formation of a peptide bond (an amide bond formed between a carboxylic acid group and an amine group) between two amino acids (the so-called “coupling” reaction). In peptide syntheses, formation of a peptide bond typically requires proper management of protecting groups, and the activation of the carboxylic acid, or a carboxyl group in general, which usually involves the use of a peptide coupling agent [for a comprehensive review on peptide coupling agents see, F. Albericio, S. A. Kates, Solid-Phase Synthesis: A Practical Guide, S. A. Kates, F. Albericio Eds; Marcel Dekker, New York, N.Y., 2000, pp. 273-328 and F. Albericio, R. Chinchilla, D. J. Dodsworth, C. Nájera, 2001, Org. Prep. Proc. Int., 33, 202].
Phosphate groups can also be activated in a way similar to the activation of carboxyl groups for coupling to amino groups in peptide synthesis. The activation of phosphate groups is an essential step in the synthesis of various nucleic acids and oligonucleotides used to build DNA and RNA, as well as molecules which mimic the chemical structure and thus the activity of the latter, which can be effected by a diverse group of activating or coupling reagents. Such activating reagents and reaction conditions which can be used for activation of phosphate groups are described in the art of peptide synthesis (see for example, L. Carpino. (1997) Methods in Enzymology, 289: 104 and WO 2006/063717) as discussed herein.
Although the synthesis of medium-large peptides for basic research is a well established procedure, the combination of the 20 naturally occurring amino acids and a growing number of unnatural amino acids makes each peptide synthesis unique at the industrial level, oftentimes requiring closer attention to each amino acid coupling. Some of the rules for coupling agents validated in the research scale can be applied at industrial level, but the results are still hardly predictable.
The two main classes of coupling techniques involve (a) those that require in situ activation of the carboxylic acid and (b) those that depend on an activated amino-acid species that has previously been prepared, isolated, purified, and characterized. The first type is by far the most convenient for the stepwise elongation of a peptide chain and is the more commonly used in convergent processes, where protected peptides are used instead of protected amino acids.
As mentioned herein, the role of the coupling agent is the activation of the carboxyl group of one amino acid which facilitates its coupling with the amino group of another amino acid. The process of activation is probably the one aspect of peptide synthesis which has been most extensively developed in recent years. An essential feature of all coupling methods is that, in addition to improving the yield of the peptide-bond formation, the configurational integrity of the carboxylic component must be maintained as well, namely no racemization should occur at any of the amino acid chiral centers.
This duality in coupling agent requirements, i.e. high peptide-bond formation yield and absence of amino acid racemization, is often difficult to achieve, since usually the most effective peptide-bond formation methods involve conversion of the acid to an intermediate bearing a good leaving group. Such leaving groups tend to increase the acidity of the α-proton of the activated amino acid, enhancing deprotonation and formation of an oxazolone, both of which lead to loss of the stereo-configuration.
Racemization is a side-reaction that occurs during the preparation of a peptide. In a production scale, the formation of small amounts of epimers can be difficult to detect and more importantly, it makes the removal of these impurities very challenging in any scale and particularly in large industrial scale processes. This constitutes one of the most serious drawbacks for the implementation of peptides as API's.
The currently most-widely used coupling reagents include carbodiimides on the one hand, and phosphonium and iminium salts on the other. It is noteworthy that coupling agents that are useful in peptide synthesis can also be used in other organic syntheses that require activation of a carboxylic moiety. Such syntheses can be used to produce organic compounds of biological interest such as, for example, peptoids, oligocarbamates, oligoamides, β-lactams, esters, polyenamides, benzodiazepines, diketopiperazines, and hydantoins.
Carbodiimides are presently the most available and low-cost coupling agents amongst the presently known reagents, and include dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) and N,N′-diisopropylcarbodiimide (DIC). The primary reactive species, O-acylisourea, is one of the most reactive species for peptide coupling. Shortcomings associated with the use of carbodiimides as coupling agents therefore mostly stem from the high and relatively uncontrollable reactivity thereof and include, for example, racemization, side reactions and low yields due to the formation of the poorly active N-acyl urea. Furthermore, while low dielectric constant solvents such as CHCl3 or CH2Cl2 are optimal for carbodiimides, the use of solvents which exhibit a higher dielectric constant such as DMF, which favor the formation of the N-acyl urea, precludes their use alone. Furthermore, dicyclohexylcarbodiimide is also incompatible with Fmoc/t-Bu solid-phase chemistry, because the urea derivative formed in such syntheses is typically not soluble in common solvents. Such urea derivatives are also difficult to remove in solution chemistry.
At the beginning of the 70's, 1-hydroxybenzotriazole (HOBt) was proposed as an additive to DCC. The addition of HOBt was aimed at reducing the racemization associated with DCC coupling. The relative success of this additive signaled the beginning of a period during which other benzotriazole derivatives such as 1-hydroxy-6-chlorobenzotriazole (6-Cl-HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) were developed and successfully used. During later years the addition of benzotriazole derivatives as additives to DCC and other carbodiimides became almost mandatory to safeguard the peptide bond formation by carbodiimide activation from low yields, undesired side reactions and loss of chirality.
In the last decade, the use of phosphonium and iminium/uronium salts, referred to herein in short as “onium salts”, of hydroxybenzotriazole derivatives as peptide coupling agents, was introduced. Although these reagents have been rapidly adopted for research purposes, only a few of them have been found compatible with current industrial requirements and synthetic strategies and were adopted by the industry. The species that reacts with onium salts is the carboxylate of the amino/organic acid. Therefore, performing the coupling reaction in the presence of at least one equivalent of a base is essential while using these reagents. The most reactive iminium salt coupling agent at present is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide (HATU).
The misconception regarding the structure of these and other coupling agents was settled by Carpino, L. A. et al. [Angew. Chem. Int. Ed. 2002, 41, No. 3, p. 441], and it is now accepted that the leaving group in these agents is linked to the iminium moiety via the triazole nitrogen and not via the oxygen, which remains in its N-oxide form. The chemical structures of HATU and analogs thereof are presented in Scheme 1 below.

XYZHATUHNPF6HBTUHCHPF6TBTUHCHBF4HCTUClCHPF6TCTUClCHBF4
Iminium salts, such as HATU, TBTU, HBTU, HCTU, or TCTU, which are possibly the most powerful coupling agents known are formed by a leaving group and a carbocation skeleton. However, the presently known peptide coupling agents are typically limited by their low desired reactivity, side products formed thereby and/or high cost.
U.S. Pat. No. 6,825,347 and WO 94/07910 teach uronium and iminium salts and their use in effecting the acylation step in amide formation, especially during peptide synthesis. The coupling agents taught in these publications have a leaving group attached to an uronium and iminium moiety, which is characterized by having N-alkyl or P-alkyl substituted nitrogen or phosphor atoms. These coupling agents, while being innovative, still provide coupling efficiencies similar to previously known coupling agents.
Phosphonium-based coupling agents are gathering grounds in the industry, and include 7-azabenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), and benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP). However, these are only marginally more effective than their carbocation/iminium counterparts and thus are oftentimes forbiddingly yet unjustifiably more expensive coupling agents. Recently, 6-chloro-1-hydroxybenzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyClock), one of the most effective phosphonium-based coupling agents, was introduced by the present assignee (see, for example, WO 2007/020620).